Crystal form of prostaglandin analogue, and preparation method and use thereof

ABSTRACT

Provided are a crystal form A of a compound having the structure as represented by formula I, and preparation method and use thereof. The X-ray powder diffraction (XRPD) chart of the crystal form A has characteristic peaks at the following diffraction angles: 2.9±0.2°, 13.6±0.2°, 17.3±0.2° and 18.6±0.2°.

TECHNICAL FIELD

This invention relates to chemical pharmaceutical fields, especially toa crystalline form of prostaglandin analogues and the preparation aswell as the use thereof.

BACKGROUND

Treprostinil (UT15) is a class of novel drugs for treating pulmonaryhypertension with the structure of formula I:

Pulmonary arterial hypertension (PAH) is a disease mainly characterizedin pulmonary arteriola vasospasm, intimal hyperplasia and remodeling.Vascular proliferation of pulmonary arteriola and remodeling will leadto progressive increase in pulmonary vascular resistance, and ultimatelyright ventricular failure and death.

Epoprostenol (Flolan) is the first prostacyclin drug approved by U.S.Food and Drug Administration (FDA) for treating PAH. Half-life ofEpoprostenol in circulation is about 3-5 mins, continuous intravenousadministration is necessary, and it should be cryopreserved beforeinfusion.

Treprostinil is an epoprostenol analog. The compound is stable underphysiological conditions, and was the first developed drug forcontinuous subcutaneous injection. Compared with intravenousadministration of epoprostenol, subcutaneous injection of treprostinilappears safer and more convenient. Inhaled formulations thereof has alsobeen approved by FDA. Treprostinil was described in U.S. Pat. No.4,306,075 for the first time. Treprostinil and other prostacyclinderivatives have been prepared according to Moriarty et al., J. Org.Chem. 2004, 69, 1890-1902, Drug of the Future, 2001, 26 (4), 364-374,U.S. Pat. Nos. 6,441,245, 6,528,688, 6,765,117, 6,809,223 and 6,756,117.

It is described in U.S. Pat. No. 5,153,222 that treprostinil isapplicable to treat pulmonary hypertension. Treprostinil was approvedfor intravenous and subcutaneous injection, and the latter avoidscontinuous intravenous catheter-related sepsis events. U.S. Pat. Nos.6,521,212 and 6,756,033 describe the treatment of pulmonaryhypertension, peripheral vascular disease, as well as other diseases andsymptoms by inhalation of treprostinil. U.S. Pat. No. 6,803,386discloses the treatment of cancer by administrating treprostinil. U.S.Patent Application Publication No. 2005/0165111 discloses the treatmentof ischemic lesions by using treprostinil. U.S. Pat. No. 7,199,157discloses the improvement of renal function by using treprostinil. U.S.Patent Application Publication No. 2005/0282903 discloses the treatmentof neuropathic foot ulcers by using treprostinil. U.S. application Ser.No. 12/028,471 discloses the treatment of pulmonary fibrosis by usingtreprostinil. U.S. Pat. No. 6,054,486 discloses the use of treprostinilfor treating peripheral vascular disease. U.S. patent application Ser.No. 11/873,645 discloses a combination therapy including treprostinil.U.S. Application Publication No. 2008/0200449 discloses the delivery oftreprostinil by using a dose-metered inhaler. U.S. ApplicationPublication No. 2008/0280986 discloses the treatment of interstitiallung disease by using treprostinil. U.S. application Ser. No. 12/028,471discloses the treatment of asthma by using treprostinil. U.S. Pat. Nos.7,417,070, 7,384,978 and U.S. Application Publication No. 2007/0078095,2005/0282901 and 2008/0249167 describe the oral dosage form oftreprostinil and other prostacyclin analogues.

In the prior art, there are few reports regarding the crystals andpurification of Treprostinil, wherein the literature (J. Org Chern 2004,69, 1890-1902) reported a ethanol-water system for recrystallization ofUT15; WO2009137066 reported a monohydrate treprostinil; and both ofcrystallization methods are similar and employ ethanol-water system. Theinventors have prepared the crude compound I according to the methodreported in the literature (J. Org. Chern. 2004, 69, 1890-1902) andobtained a slightly yellow gummy solid, however, the reaction liquidsare difficult to be filtered and the residual solvent can not becompletely removed under reduced pressure. Then, the obtained productwas recrystallized using ethanol-water system, and dried under reducedpressure to give an extremely viscous substance. Such substance remainsviscous after being stored at low-temperature (−20° C. to 0° C.), and nosolidification or crystallization occurs at all.

Generally, prostaglandins have poor stability, and should be storedbelow −20° C. In view of the stability and purity of the compound, thereis an urgent need in the art to obtain a stable crystal form of compoundI.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel and stablecrystalline form of compound I.

Another purpose of the present invention is to provide a preparationmethod for the novel crystalline form of compound I.

Another purpose of the present invention is to provide the use of thenovel crystalline form of compound I.

The fourth purpose of the present invention is to provide apharmaceutical composition comprising the novel crystalline form ofcompound I.

In the first aspect of the present invention, crystalline form A of acompound is provided, wherein the compound has the structure of formulaI, and said crystalline form A has characteristic peaks at the following2θ angles in the X-ray Powder diffraction (XRPD) pattern: 2.9±0.2°,13.6±0.2°, 17.3±0.2° and 18.6±0.2°;

Preferably, said crystalline form A may has other characteristic peak atthe following 2θ angle in the X-ray powder diffraction pattern:21.8±0.2°.

The maximum peak in the differential scanning calorimetry (DSC) for thecrystalline form A of the compound provided by the invention exists at123° C.-128° C.; preferably, at 125° C.; and more preferably, thedifferential scanning calorimetry (DSC) for the crystalline form A isshown as in FIG. 2.

Infrared Spectrum of said crystalline form A of the compound provided bythe invention is shown in FIG. 3.

In the second aspect of the present invention, a preparation method forthe crystalline form A of compound I is provided, comprising thefollowing steps:

(1) the crude compound of formula I is mixed with one or more solventsselected from following solvent 1 to obtain solution 1: methanol,acetonitrile, ethyl acetate, methyl acetate, isopropyl acetate, andt-butyl acetate;

(2) solution 1 is stirred and cooled, or solvent 2 is added to give thecrystalline form A of compound I provided by the invention, wherein saidsolvent 2 is selected from one or more of the following: water, C5-C8alkane.

In step (1) of the above preparation method, the temperature for mixingis at 0-80° C.; preferably, at 20-50° C.; more preferably, at 30-50° C.

In step (1) of the above preparation method, the weight to volume ratiofor mixing the crude compound of formula I and solvent 1 is 1:0.5-50(g:ml); preferably, 1:0.5-20 (g:ml); and more preferably, 1:1-5 (g:ml).

In step (2) of the above preparation method, the temperature is cooledto −25° C. to 25° C.; preferably, to −5° C. to 5° C.; and morepreferably, to −2° C. to 2° C.

In step (2) of the above preparation method, after cooled, solution 1 isstirred for 5-50 hours; preferably, 10-30 hours; and more preferably,12-25 hours.

In the above preparation method, the volume ratio of solvent 2 in steps(2) to solvent 1 in step (1) is 0.1-1:1; preferably, 0.2-0.8:1; and morepreferably, 0.4-0.6:1.

In step (2) of the above preparation method, after solvent 2 is added,the resulting mixture is cooled to 1 to 10° C., and then is stirred for5-50 hours; and preferably, cooled to 3 to 7° C., and then is stirredfor 10-30 hours.

In the third aspect of the present invention, a pharmaceuticalcomposition is provided, said pharmaceutical composition comprisingcrystalline form A of compound I provided by the invention and apharmaceutically acceptable carrier.

In the fourth aspect of the present invention, the use of crystallineform A of compound I as said above is provided, for preparingpharmaceutical composition for treating pulmonary arterial hypertensionand peripheral vascular disease.

Based on the above, a stable crystalline form of compound I is obtainedby the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray powder diffraction pattern of crystalline form Aof compound I obtained in Examples of the invention;

Peak 2-θ d (A) I % (Relative Intensity) 1 2.960 29.8272 100 2 6.52013.5453 10.3 3 13.520 6.5437 22.8 4 17.319 5.1159 24.5 5 18.581 4.771326.6 6 21.722 4.0880 18.3

FIG. 2 is the differential scanning calorimetry (DSC) of crystallineform A of compound I obtained in Examples of the invention.

FIG. 3 is the Infrared Spectrum (IR) of crystalline form A of compound Iobtained in Examples of the invention.

FIG. 4 shows the X-ray powder diffraction pattern of crystalline form ofcompound I obtained in Comparative Example of the invention;

MODES FOR CARRYING OUT THE INVENTION

Upon research, the inventors have discovered that a single crystallineform A of compound I can be obtained by: dissolving compound I intomethanol, acetonitrile, ethyl acetate, methyl acetate, isopropylacetate, and t-butyl acetate or the combinations thereof to form ahomogeneous solution, and then performing crystallization and changingsuch factors as the temperature of crystallization, mol concentration,cooling rate or stirring speed. Alternatively, a single crystalline formA of compound I can be obtained by: dissolving compound I into methanol,acetonitrile, ethyl acetate, methyl acetate, isopropyl acetate, andt-butyl acetate or the combinations thereof to form a homogeneoussolution, diluting the solution of compound I using a poor solvent, suchas pentane, n-hexane, cyclohexane, heptane, or the combinations thereofor water, performing crystallization and changing such factors as thetemperature of crystallization, mol concentration, cooling rate andstirring speed, or crystallization time. Crystalline form A is a stablecrystalline form, and high yield of crystallization can be obtained bysuch method.

As used herein, the chemical formula or name shall include all opticalisomers and stereoisomers as well as racemic mixtures comprising theseisomers and mixtures thereof.

Crystalline Form A of Compound I

Upon research, the inventors have surprisingly discovered that a singlecrystalline form A of compound I can be obtained by mixing compound Iwith a polar solvent to form a homogeneous solution, and then changingsuch factors as the temperature of crystallization, mol concentration,stirring speed; alternatively, by mixing compound I with a polar solventto form a homogeneous solution, diluting the resulting solution using apoor solvent, and then changing such factors as the temperature ofcrystallization, mol concentration, stirring speed, which demonstratesthat crystalline form A is a unique advantageous crystalline form. Then,a sample of compound I with low residual solvent, even without residualsolvent, can be obtained by simple drying methods. Therefore, the defectof excess of residual solvent, and the difficulties in sampling as wellas split charging can be overcome. Moreover, the inventors havesurprisingly discovered that crystalline form A of compound I has aunique stability which is favorable to preservation.

In the present invention, crystalline form A of the compound of formulaI is provided, and the properties thereof were studied using a varietyof methods and instruments.

X-ray powder diffraction, namely X-ray polycrystal diffraction, is acommonly used experimental method for determining the crystallinestructure (i.e., crystalline form). A series of X-ray diffractionpatterns can be produced by using X-ray powder diffractometer, whenX-ray passing through a crystal. In the pattern, different diffractionlines and the intensities thereof are determined by atomic clusterhaving certain structure. Therefore the specific structure of acrystalline form can be determined.

The methods for determining the X-ray diffraction pattern of acrystalline form are known in the art. For example, the pattern can beobtained by using Bruker D8 Advanced X-ray powder diffractometer withthe scanning rate of 2°/min and copper irradiated target being used.

The crystalline form A of compound I according to the present inventionpossesses the unique crystal form and specific characteristic peaks inthe X-ray powder diffraction (XRPD) pattern. Particularly, thecrystalline form A of compound I according to the present inventionpossesses characteristic peaks at the following 2θ angles in the X-raypowder diffraction (XRPD) pattern:2.9±0.2°, 13.6±0.2°, 17.3±0.2° and18.6±0.2°; and preferably, further possesses characteristic peak at thefollowing 2θ angle: 21.8±0.2°. Most preferably, said crystalline form Apossesses the X-ray powder diffraction (XRPD) pattern substantiallyidentical with FIG. 1.

“Differential scanning calorimetry” (DSC) is a technology for measuringthe relationship of energy difference and temperature between the testedsubstance and the reference during the heating process. On the DSCpattern, the location, form and number of the peak are relevant to theproperties of the substance; therefore, the substance can bequalitatively identified by using DSC. Said method is used in the art todetect many parameters of a substance, such as the phase transitiontemperature, glass transition temperature and reaction heat.

DSC is known in the art. For example, DSC pattern of a crystal can beobtained by using DSC Q20 differential scanning calorimeter under thefollowing conditions: warming rate of 10° C./min, from 25° C. to 200° C.

In one embodiment of the present invention, the crystalline form A ofcompound I obtained by the method according to the present invention wasdetermined to have the maximum peak at 120-130° C. by DSC; preferably,at 124.84° C.; and more preferably, the crystalline form A of compound Iaccording to the present invention has the DSC pattern substantiallyidentical with FIG. 2.

The crystal structure can also be determined by Infrared Spectrometry(IR), which is known in the art. For example, it can be determined byusing PE Spectrum One B, tableting at KBr: sample=200:1, and scanning400˜4000 cm⁻¹. The crystalline form A of compound I according to thepresent invention has characteristic peaks at the following wavenumbers: 3432.44 cm⁻¹, 3380.48 cm⁻¹, 3307.00 cm⁻¹, 2929.82 cm⁻¹,2854.72cm⁻¹, 2590.52 cm⁻¹, 1936.53 cm⁻¹, 1740.01 cm⁻¹, 1711.90 cm⁻¹,1607.45 cm⁻¹, 1584.91 cm⁻¹, 1469.58 cm⁻¹, 1423.93 cm⁻¹, 1327.64cm⁻¹,1310.81 cm⁻¹, 1258,89 cm⁻¹, 1147.72 cm⁻¹, 1122.09 cm⁻¹, 1082.36 cm⁻¹,1026.45 cm⁻¹, 890.36 cm⁻¹, 788.35 cm⁻¹, 776.19 cm⁻¹, 737.98 cm⁻¹.Preferably, the crystal has the IR pattern substantially identical withFIG. 3.

Preparation Method for Crystalline Form A of Compound I

The present invention also provides a method for preparing crystallineform A of the compound of formula I.

In one embodiment provided in the present invention, a preparationmethod for the crystalline form A of compound I comprises the followingsteps:

(1) the crude compound of formula I is mixed with solvent 1 to obtainsolution 1; and solvent 1 is selected from the following: methanol,acetonitrile, ethyl acetate, methyl acetate, isopropyl acetate, and/ort-butyl acetate;

(2) solution 1 is cooled or solvent 2 is added and stirred to give thecrystalline form A of compound I.

In step (1), the mixing should be carried out below the boiling point ofsolvent 1, preferably at 0-80° C.; more preferably, at 20-50° C.; morepreferably, at 30-50° C.

In step (1), the ratio (weight to volume) for mixing the crude compoundof formula I and solvent 1 is 1:0.5-50 (g:ml); preferably, 1:0.5-20;more preferably, 1:1-5 (g:ml).

In step (1), solution 1 is a homogeneous solution obtained by thoroughlymixing the crude compound of formula I with solvent 1 and dissolving thecompound into solvent 1.

In step (2), the temperature should not be lowered to below the meltingpoint of solution 1, which can be lower than the temperature for mixingin step (1) by 25-80° C.; and preferably, can be lowered to −25° C. to25° C.; more preferably, to −5° C. to 5° C.; and more preferably, to −2°C. to 2° C.

Solvent 2 can be one or more selected from the following group: water,and C5-C8 alkane; preferably, selected from n-hexane, n-heptane,cyclohexane, methyl tert-butyl ether and/or petroleum ether.

The volume ratio of solvent 2 in steps (2) to solvent 1 in step (1) is0.1-1:1; preferably, 0.2-0.8:1; and more preferably, 0.4-0.6:1.

In step (2), after solvent 2 is added, the resulting mixture is cooledto 1 to 10° C., and then is stirred for 5-50 hours; and preferably,cooled to 3 to 7° C., and then is stirred for 10-30 hours.

More preferably, in said embodiment, step (3) can be included posteriorto step (2), the crystalline form A of compound 1 is filtered, thefilter cake is washed by using solvent 1 and dried under reducedpressure to give a solid of crystalline form A of compound I.

In step (3), washing can be performed according to routine methods inthe art, the purpose for which is to remove a few impurities, i.e.,remove impurities adhering to the surface of crystals by washing.

Use of Crystalline Form A of Compound I

Crystalline form A of compound I provided by the present invention hasgood stability, and can be used as raw material medicament for thetreatment of pulmonary hypertension and peripheral vascular disease.

The medicament comprises crystalline form A of the compound of formula Iand a pharmaceutically acceptable carrier. Depending on the route ofadministration, the medicament can be prepared into various dosageforms. The dosage forms can be administered through one of the followingroutes: oral, inhalation, transdermal, etc. Moreover, it is necessary tobe noted that the dosage and methods of use of crystalline form A ofcompound I according to the present invention depend on various factors,including the age, weight, gender, natural health status, nutritionalstatus of the patient, intensity of activity of compound,administration, metabolic rate, the severity of the disease andattending physician's subjective judgment.

As used herein, “crude compound of formula I” means compound of formulaI in amorphous form prepared by the methods known in the art.Preferably, the crude compound I without purification can be prepared bythe method reported in J. Org. Chern. 2004, 69, 1890-1902, and using(1R, 2R, 3aS,9aS)-2,3,3a,4,9,9a-hexahydro-1-[(3S)-3-hydroxyoctyl]-1H-phenyl[f]indene-2,5-diolas a starting material.

As used herein, “HPLC purity” refers to the percentage of the peak areaof compound I obtained by area normalization method in the sum of allthe peak area in HPLC detection applied to compound I, according to theobtained chromatogram.

Column: 4.6×250 mm 5 μm C18 column; flow rate: 2 ml/min; injectionvolume: 10 μl; column temperature: 35° C.; detection wavelength: 217 nm;mobile phase: acetonitrile/water/trifluoroacetic acid; detection limit:10 ng.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier for the administration of therapeutic agents, includingvarious excipients and diluents. This term refers to such pharmaceuticalcarriers that they are not necessary active ingredients, and won't causeundue toxicity upon administration. Suitable carriers are well-known tothe skilled person in the art. Detailed discussion on thepharmaceutically acceptable excipients can be found in Remington'sPharmaceutical Sciences (Mack Pub. Co., NJ 1991). In a composition, apharmaceutically acceptable carrier may include a liquid, such as water,saline, glycerol and ethanol. Moreover, there may be auxiliarysubstances, such as disintegrants, wetting agents, emulsifying agents,pH buffering substances and the like in these carriers.

All the features mentioned above or in the examples below of theinvention can be optionally combined. All features disclosed in thisspecification may be used in any combination. Any alternative featureserving the same, equivalent, or similar purpose may replace eachfeature disclosed in this specification. Therefore, unless otherwisespecified, the features as disclosed are only general examples ofequivalent or similar features.

The main advantages of the invention include:

1. Crystalline form A of the present invention has a unique stability,

2. The defect of cryopreservation for compound I can be overcome basedon the specific crystalline form provided by the present invention.

The invention will be further illustrated with reference to thefollowing specific examples. It is to be understood that these examplesare only intended to illustrate the invention, but not to limit thescope of the invention. For the experimental methods in the followingexamples without particular conditions, they are performed under routineconditions or as instructed by the manufacturer. Unless otherwisespecified, all percentages, ratios, proportions or parts are by weight.

The unit of the weight/volume percentages in the invention is well knownto the skilled in the art, for example, the weight of a solute in a 100mL solution.

Unless otherwise defined, all scientific and technical terms used hereinhave the same meaning as commonly understood by the skilled in the art.Furthermore, any process or material similar or equivalent to thosedescribed herein can be used in the process of the present invention.The preferred embodiments and materials described herein are merelyprovided for illustration.

HPLC detection method mentioned in the following Examples are listed asfollows:

Column: 4.6×250 mm 5 μm C18 column; flow rate: 2 ml/min; injectionvolume: 10 μl; column temperature: 35° C.; detection wavelength: 217 nm;mobile phase: acetonitrile/water/trifluoroacetic acid; Elutioncondition: water (60%): acetonitrile (40%): trifluoroacetic acid (0.1%);detection limit: 10 ng.

The purity mentioned in the following Examples is HPLC purity.

Example 1

Preparation of the Crude Compound I

The crude compound I (41 g) without purification can be prepared by themethod reported in J. Org. Chern. 2004, 69, 1890-1902, and using(1R,2R,3aS,9aS)-2,3,3a,4,9,9a-hexahydro-1-[(3S)-3-hydroxyoctyl]-1H-phenyl[f]indene-2,5-diolas a starting material.

Comparable Example 2

1.0 g of the crude compound was crystallized in ethanol/water (1:1)according to the method reported in J. Org. Chern. 2004, 69, 1890-1902,to give a gummy solid (0.71 g). The purity of solid is 99.23%, and theX-ray powder diffraction pattern thereof is shown in FIG. 4.

Comparable Example 3

1.0 g of the crude compound was crystallized and dried according to themethod reported in WO2009137066 to give a gummy solid (0.73 g). Thepurity of solid is 99.39%.

Example 4

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and methanol (1.0 ml). The temperature wasincreased to 30° C. for dissolving the crude compound to give ahomogeneous solution. Pure water (0.6 ml) was slowly added. The solutionwas cooled to 5° C. and stirred for 10 h. The precipitate was filteredoff, washed by 10% aqueous solution of methanol for 2-3 times at 5° C.,and dried to give 0.91 g of crystalline solid. For the solid, X-raypowder diffraction pattern is consistent with FIG. 1, DifferentialScanning calorimetry (DSC) is consistent with FIG. 2, and InfraredSpectrometry is consistent with FIG. 3, and HPLC purity is 99.90%.Organic residue: methanol 0.04% (yield: 91%).

Example 5

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and ethyl acetate (5.0 ml). Thetemperature was increased to 40° C. for dissolving the crude compound togive a homogeneous solution. And the solution was cooled to 0° C. andstirred for 12 h. The precipitate was filtered off, washed by ethylacetate for 2-3 times at 0° C., and dried to give 0.85 g of crystallinesolid. For the solid, X-ray powder diffraction pattern is consistentwith FIG. 1, Differential Scanning calorimetry (DSC) is consistent withFIG. 2, and Infrared Spectrometry is consistent with FIG. 3, and HPLCpurity is 99.74%. Organic residue: ethyl acetate 0.03% (yield: 85%).

Example 6

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and ethyl acetate (4.0 ml). Thetemperature was increased to 40° C. for dissolving the crude compound.N-hexane (2 ml) was added at the same temperature dropwise. And theresulting mixture was cooled to 3° C. and stirred for 20 h. Theprecipitate was filtered off, washed by ethyl acetate and n-hexane (1:1)for 2-3 times at 5° C., and dried to give 0.98 g of crystalline solid.For the solid, X-ray powder diffraction pattern is consistent with FIG.1, Differential Scanning calorimetry (DSC) is consistent with FIG. 2,and Infrared Spectrometry is consistent with FIG. 3, and HPLC purity is99.50%. Organic residue: ethyl acetate 0.04%, n-hexane 0.01% (yield:98%).

Example 7

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and ethyl acetate (3.0 ml). Thetemperature was increased to 50° C. for dissolving the crude compound togive a homogeneous solution. N-heptane (1.2 ml) was added at the sametemperature dropwise. And the resulting mixture was slowly cooled to 7°C. and stirred for 30 h. The precipitate was filtered off, washed byethyl acetate and n-hexane (2:1) for 2-3 times at 5° C., and dried togive 0.68 g of crystalline solid. For the solid, X-ray powderdiffraction pattern is consistent with FIG. 1, Differential Scanningcalorimetry (DSC) is consistent with FIG. 2, and Infrared Spectrometryis consistent with FIG. 3, and HPLC purity is 99.80%. (yield: 68%).

Example 8

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and methyl acetate (3.5 ml). Thetemperature was increased to 40° C. for dissolving the crude compound togive a homogeneous solution. And the solution was slowly cooled to −2°C. and stirred for 25 h. The precipitate was filtered off, washed bymethyl acetate for 2-3 times at 0° C., and dried to give 0.84 g ofcrystalline solid. For the solid, X-ray powder diffraction pattern isconsistent with FIG. 1, Differential Scanning calorimetry (DSC) isconsistent with FIG. 2, and Infrared Spectrometry is consistent withFIG. 3, and HPLC purity is 99.80% (yield: 84%).

Example 9

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and isopropyl acetate (3.0 ml). Thetemperature was increased to 50° C. for dissolving the crude compound togive a homogeneous solution. Cyclohexane (0.6 ml) was added at the sametemperature dropwise. And the resulting mixture was slowly cooled to 5°C. and stirred for 30 h. The precipitate was filtered off, washed byisopropyl acetate and cyclohexane (2:1) for 2-3 times at 5° C., anddried to give 0.72 g of crystalline solid. For the solid, X-ray powderdiffraction pattern is consistent with FIG. 1, Differential Scanningcalorimetry (DSC) is consistent with FIG. 2, and Infrared Spectrometryis consistent with FIG. 3, and HPLC purity is 99.50% (yield: 72%).

Example 10

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and ethyl acetate (4.0 ml). Thetemperature was increased to 40° C. for dissolving the crude compound togive a homogeneous solution. Methyl tert-butyl ether (0.8 ml) was addedat the same temperature dropwise. And the resulting mixture was slowlycooled to −5° C. and stirred for 20 h. The precipitate was filtered off,washed by ethyl acetate and methyl tert-butyl ether (2:1) for 2-3 timesat 5° C., and dried to give 0.86 g of crystalline solid. For the solid,X-ray powder diffraction pattern is consistent with FIG. 1, DifferentialScanning calorimetry (DSC) is consistent with FIG. 2, and InfraredSpectrometry is consistent with FIG. 3, and HPLC purity is 99.55%(yield: 86%).

Example 11

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (1.0 g) and t-butyl acetate (3.0 ml). Thetemperature was increased to 50° C. for dissolving the crude compound togive a homogeneous solution. Petroleum ether (1.2 ml) was added at thesame temperature dropwise. And the resulting mixture was slowly cooledto 5° C. and stirred for 30 h. The precipitate was filtered off, washedby t-butyl acetate and petroleum ether (2:1) for 2-3 times at 5° C., anddried to give 0.80 g of crystalline solid. For the solid, X-ray powderdiffraction pattern is consistent with FIG. 1, Differential Scanningcalorimetry (DSC) is consistent with FIG. 2, and Infrared Spectrometryis consistent with FIG. 3, and HPLC purity is 99.62% (yield: 80%).

Example 12

Preparation of Crystalline Form A of Compound I

Into a 25 ml eggplant shaped bottle, was added the crude compound Iobtained in Example 1 (0.1 g) and acetonitrile (4.5 ml). The temperaturewas increased to 50° C. for dissolving the crude compound to give ahomogeneous solution. And the solution was slowly cooled to 2° C. andstirred for 10 h. The precipitate was filtered off, washed byacetonitrile for 2 times at 0° C., and dried to give 0.85 g ofcrystalline solid. For the solid, X-ray powder diffraction pattern isconsistent with FIG. 1, Differential Scanning calorimetry (DSC) isconsistent with FIG. 2, and Infrared Spectrometry is consistent withFIG. 3, and HPLC purity is 99.60% (yield: 85%).

Example 13

Stability Comparison

Compound I from example 2, example 3 and example 4 were obtained, andsubjected to stability test at 40° C., respectively. The HPLC resultsdemonstrated that crystalline form A of compound I did not degrade atall, after stored at 0° C. for 12 months, at 20° C. for 6 months, or at40° C. for 2 months. Therefore, crystalline form A of compound Iprepared by the method of the present invention has unique stability.

Purity Purity Crystalline Purity at 0 day at 10 day Purity at 30 day at90 day Form (40° C.) (40° C.) (40° C.) (40° C.) Exampl 2 99.23% 99.23%99.15% 98.63% Example 3 99.39% 99.38% 99.31% 99.09% Example 4 99.90%99.91% 99.89% 99.88%

Example 14

Pharmaceutical Composition

Crystalline form A of compound I prepared in Example 2 (17.4 mg), sodiumchloride (189 mg), sodium citrate (183 mg), sodium hydroxide (5.8 mg),and 1 N HCl (117 mg) were mixed into 20 ml of pure water. And then purewater was supplemented to the final volume of 29 ml. The mixture wasdivided into 10 aliquots, and the aliquots were added into 3 ml ampoulebottles (n=10), respectively. Each ampoule was loaded into a nebulizerfor treating pulmonary hypertension or peripheral vascular disease byinhalation.

The above examples are merely the preferred examples for the presentinvention, and such examples cannot be used to limit the scope of theinvention. The substantial technical contents according to the presentinvention are broadly defined in the claims. And any entities or methodsaccomplished by others should be considered as the equivalents and fallwithin the scope as defined by the claims, if said entities or methodsare the same as those defined by the claims.

What we claimed is:
 1. Crystalline form A of a compound, wherein thecompound has the structure of formula I, and said crystalline form A hascharacteristic peaks at the following 2θ angles in the X-ray Powderdiffraction (XRPD) pattern: 2.9±0.2°, 13.6±0.2°, 17.3±0.2° and18.6±0.2°;


2. The crystalline form A of the compound according to claim 1, whereinsaid crystalline form A may has other characteristic peak at thefollowing 2θ angle in the X-ray powder diffraction pattern: 21.8±0.2°.3. The crystalline form A of the compound according to claim 1, whereinthe maximum peak in the differential scanning calorimetry (DSC) for saidcrystalline form A exists at 123° C. -128° C.; and preferably, at 125°C.
 4. The crystalline form A of the compound according to claim 3,wherein the differential scanning calorimetry (DSC) for said crystallineform A is shown as in FIG.
 2. 5. The crystalline form A of the compoundaccording to claim 1, wherein Infrared Spectrum of said crystalline formA is shown in FIG.
 3. 6. A preparation method for crystalline form A ofcompound I according to claim 1, comprising the following steps: (1) thecrude compound of formula I is mixed with one or more solvents selectedfrom following solvent 1 to obtain solution 1: methanol, acetonitrile,ethyl acetate, methyl acetate, isopropyl acetate, and t-butyl acetate;(2) solution 1 is stirred and cooled, or solvent 2 is added to give thecrystalline form A of compound I according to claim 1, wherein saidsolvent 2 is selected from one or more of the following: water, andC5-C8 alkane.
 7. The preparation method according to claim 6, wherein,in step (1), the temperature for mixing is at 0-80° C.; preferably, at20-50° C.; more preferably, at 30-50° C.
 8. The preparation methodaccording to claim 6, wherein, in step (1), the weight to volume ratiofor mixing the crude compound of formula I and the solvent 1 is 1:0.5-50(g:ml); preferably, 1:0.5-20 (g:ml); more preferably, 1:1-5 (g: ml). 9.The preparation method according to claim 6, wherein, in step (2), thetemperature is cooled to −25° C. to 25° C.; preferably, −5° C. to 5° C.;more preferably, −2° C. to 2° C.
 10. The preparation method according toclaim 6, wherein, in step (2), after cooled, the solution is stirred for5-50 hours; preferably, 10-30 hours; and more preferably, 12-25 hours.11. The preparation method according to claim 6, wherein, the volumeratio of solvent 2 in steps (2) to solvent 1 in step (1) is 0.1-1:1;preferably, 0.2-0.8:1; more preferably, 0.4-0.6:1.
 12. The preparationmethod according to claim 6, wherein, in step (2), after solvent 2 isadded, the resulting mixture is cooled to 1 to 10° C., and then isstirred for 5-50 hours; and preferably, cooled to 3 to 7° C., and thenis stirred for 10-30 hours.
 13. A pharmaceutical composition, whereinsaid pharmaceutical composition comprises crystalline form A of compoundI according to claim 1 and a pharmaceutically acceptable carrier.